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C-MYC, BCL2 and BCL6 Translocation in B-Cell Non-Hodgkin Lymphoma Cases

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C-MYC, BCL2 and BCL6 Translocation in B-Cell Non-Hodgkin Lymphoma Cases Journal of Cancer 2020, Vol. 11 190 Ivyspring International Publisher Journal of Cancer 2020; 11(1): 190-198. doi: 10.7150/jca.36954 Research Paper C-MYC, BCL2 and BCL6 Translocation in B-cell Non-Hodgkin Lymphoma Cases Dayang Sharyati Datu Abdul Salam1, Ei Ei Thit2, Siew Hoon Teoh1, Soo Yong Tan3, Suat Cheng Peh4, Shiau-Chuen Cheah1 1. Faculty of Medicine and Health Sciences, UCSI University, Kuala Lumpur, Malaysia. 2. Advanced Molecular Pathology Laboratory, SingHealth Tissue Repository, Singapore. 3. National University Hospital, Singapore. 4. Sunway University Hospital, Selangor, Malaysia. Corresponding author: [email protected] © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.05.23; Accepted: 2019.09.23; Published: 2020.01.01 Abstract C-MYC, BCL2 and BCL6 genes are the most commonly oncogenes involved in B-Cell lymphomas. Translocations of these oncogenes are associated with an aggressive clinical course. This study aims to elucidate the patterns of BCL6, BCL2 and C-MYC gene aberrations among Malaysian B-cell Non-Hodgkin Lymphoma (NHL) using fluorescence in situ hybridization (FISH). Eighty-one B-cell NHL tissue blocks were retrieved between the year 2011 to 2015 and investigated using immunohistochemistry and interphase FISH dual colour break-apart probes of BCL2, BCL6, C-MYC and IgH. A significant difference was detected between the nodal and extranodal sites in all the BCL2 (p=0.01), C-MYC (p=0.03) and IgH (p=0.006) cases except for BCL6 (p=0.2). Our study showed that BCL6 had the highest gene translocation while BCL2/BCL6 had the most mixed aberrations of gain copies and translocation, however no mixed aberrations of gain copies and translocation was found in C-MYC. None of the mixed gain copies and translocation was found in any of the germinal centre B-cell (GCB) subtype of Diffuse Large B-cell Lymphoma, however, five were found in BCL6 and IgH gene in the non-GCB subtype; while mixed gain copies and translocation cases of BCL2 gene was found in the Follicular Lymphoma cases only. The study found interesting findings of BCL2, C-MYC and IgH gene aberrations between nodal and extranodal sites. This information might benefit future study in predicting prognosis and determine effective therapeutic strategies in the multi-ethnic populations of Malaysia as well as the Asian population. Key words: B-cell Non-Hodgkin Lymphoma, C-MYC, BCL2, BCL6, gene abberation, FISH Introduction There has been an intense upsurge worldwide diagnosing cancer cell type, thus leads to lesser for Non-Hodgkin lymphoma (NHL) incidence misdiagnosis cases of NHL as well as better approximately 2-4% annually since the last seventy classification of gray-zone lymphomas [4]. In years [1]. Based on the statistics presented, it was in comparison to western countries, Asian countries accordance to the study done by Devesa and Fears [2] have lower median age of NHL. The representative whereby western countries have the highest increased median age of patient in western countries was 68 observed. Both males and females have seen an years old [5]. However, the median age of NHL increased in all age groups however racial differences patient in Asian countries such as Iran, Korea and was observed in age-specific incidence curves up until Taiwan are between 50-55 years old [6-8]. In the the age of 45 and 35 for male and female respectively Global Cancer Facts 3rd Edition [9], the global trend [3]. The reasons for the increase include wider use of suggested that the increase of NHL cases in most immunohistochemical method for screening and developed countries reflects a true increase in disease http://www.jcancer.org Journal of Cancer 2020, Vol. 11 191 occurrence. However, improvements in diagnostic in cell cycle control, proliferation and differentiation, methods and modification of classification may also apoptosis, and DNA damage response. Loss of play a role in the increase of NHL cases [10]. normal control mechanisms regulating BCL6 Double-hit translocation refer to translocation of expression causes lymphoproliferative disease, C-MYC with another concurrent breakpoints such as resembling DLBCL. BCL6 translocation, t(3q27), is BCL2, BCL6, BCL1 or BCL3. Triple-hit translocation responsible for up to 35% of DLBCL cases, the largest consists of C-MYC, BCL2 and BCL6. Currently, these compared to other DLBCL gene translocations [17]. gene translocations do not have any baseline Deregulation of BCL6 located on chromosome 3 is characteristics aside from the genetic profile that can believed to contribute to malignant transformation in be confirmed through various genetic tests such as germinal center–derived B cells (GCB). Detection of fluorescence in situ hybridisation (FISH). Also, point mutations of the regulatory region of the BCL6 morphologically they are difficult to distinguish and gene have been frequently found in GCB and post– are usually unclassifiable B-cell lymphoma of GCB lymphomas, including FL, DLBCL, and BL [18]. intermediate characteristic between aggressive The genetic aberrations that are representative of lymphoma, Diffuse Large B-cell Lymphoma (DLBCL), specific subtypes of NHL mentioned previously can and very aggressive lymphoma, Burkitt Lymphoma be identified by genetic diagnosis. Particularly, the (BL). use of FISH studies to diagnose NHL is becoming C-MYC translocation of t(8;14)(q24;q32) usually increasingly important. Interphase FISH is the most deregulate C-MYC expression. Rearrangement of the commonly applied technique for the demonstration of C-MYC gene situated on chromosome 8 next to the gene translocation, as it can be applied on paraffin IgH gene, or lambda (λ) and kappa (κ) light chain embedded tissue, enabling studies on archival genes subsequently caused upregulation of gene material. In comparison to immunohistochemical expression commonly observed in BL. In normal cells, staining, interphase FISH are able to visualize gene C-MYC acts as a transcriptional regulator involved expression pattern and can provide spatial and largely with cell cycle progression (from G1 to S temporal information on understanding gene function phase) and the inhibition of terminal differentiation. which immunohistochemistry are not able to provide. Over-expression in normal cells sensitizes the cell to a Hence, the study aims to characterize the patterns of variety of apoptotic triggers which leads the cell to BCL6, BCL2 and C-MYC gene aberrations in resist cell death ultimately result in cancer. C-MYC Malaysian B-cell NHL using interphase FISH. gene rearrangement is reported to be rare occurrence in Follicular Lymphoma (FL). However, it was Patients and Methods observed in 7-15% of de novo DLBCL [11] and 8% of Cases selection post-transformation DLBCL [12]. Several studies reported [13,14] the presence of C-MYC gene A total of 81 B-cell Non-Hodgkin Lymphoma rearrangement upon transformation. Hence, it is (NHL) were retrieved from a private hospital in highly probable that C-MYC plays a vital role in Penisular Malaysia laboratory, the Pantai Premier transformation of high grade B-cell malignancies. Pathology Malaysia archives between the years 2011 BCL2 translocation, t(14;18)(q32;q21), is observed to 2015. The demographic data of these patients, in about 15-20% of DLBCL cases and approximately in immunohistochemistry slides and tissue blocks were 80-90% of FL cases [15]. BCL2 came from the BCL obtained from the Pantai Premier Pathology database family proteins which has a crucial role in cell by the referring clinician. All the B-cell NHL were apoptosis. BCL2 function as a pro-survival protein, sectioned at 3 µm for immunohistochemical staining represses apoptotic cell death, and protects cell from a using a panel of monoclonal and polyclonal wide range of cytotoxic insults including cytokine antibodies according to Pantai Premier Pathology’s deprivation, ultraviolet irradiation. Deregulation of standard operating procedure. For the DLBCL BCL2 leads to over-expression of BCL2, making the classification of GCB and non-GCB subtype, a panel of cell to resist cell death. In contrast, BCL2 inhibition three antigens namely CD10, BCL6 and MUM1 was eliminate cell survival advantage and allow apoptosis used according to Hans Criteria [19]. to occur. Similar to C-MYC translocation characteristic Morphology in DLBCL, chromosomal rearrangement that mix the The materials were cut into 4 µm thick sections BCL2 gene situated on chromosome 18 involving the and stained with haematoxylin-eosin for histologic IgH gene deregulates the BCL2 expression [16]. evaluation. All specimens were reviewed by a Coming from the same BCL family proteins, pathologist (PSC) for confirmation and classification BCL6 is essential for the development of germinal according to the 2008 WHO Classification of tumours centre in B-cells. It acts as a transcriptional repressor of haematopoietic and lymphoid tissues [4] using http://www.jcancer.org Journal of Cancer 2020, Vol. 11 192 Leica DM500 light microscope (Leica Microsystems, Lymphoid Tissue (MALT) cases (1%), 1 Mediastinal Switzerland) graded reticule at x10 and x40 B-cell Lymphoma case (1%) and 1 magnification. DLBCL-Plasmablastic Lymphoma case (1%). In terms of lymphoma sites, B-cell lymphoma originating from Interphase FISH and scoring nodal sites was 42 (52%) and extranodal sites was 39 A total of 81 cases were examined using four (48%). The most common extranodal sites are interphase FISH dual colour break-apart probes gastrointestinal (12 cases, 31%) and Waldeyer’s ring (8 namely BCL2/18q21, BCL6/3q27, C-MYC/8q24 and cases, 21%) and 19 others for example in the brain or IgH/14q32 (Dako, Denmark). Haematoxylin-eosin spleen. stained tissue sections from representative formalin-fixed, paraffin-embedded tissue blocks were Table 1. Demographic and clinical characteristic of 81 patients used to define the tumour areas. FISH analysis was with B-Cell NHL performed on 2 µm tissue sections using FISH DNA break-apart probes and FISH Ancillary Kit (Vysis N (%) Gender probe, Abbott, United States).
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